Unless matched to oncogenic driver mutations through companion diagnostics, clinical trials with targeted drugs are mostly unsuccessful. There is a fundamental gap between the discovery of active targeted drugs and the identification of mechanistic biomarkers needed to utilize their full clinical potential. Filling tis gap is imperative, because it will produce new personalized medicine opportunities for patients who currently have limited therapeutic options. Our long-term goal is to contribute to the improvement of personalized medicine by elucidation of the complex mechanisms of action (MoAs) of targeted drugs in lung cancer thereby revealing targets and pathways with clinical significance. Many targeted drugs, particularly kinase inhibitors, are pleiotropic and their anticancer activity can result from non-obvious, off-target effects. Importantly, our preliminary data strongly indicate beneficial off-target activity of several clinically relevant kinase inhibitrs, namely tivantinib, midostaurin and crizotinib, in non-small cell lung cancer (NSCLC) cells. The objective of this project is to identify the molecular MoAs that underlie these off-target effects n NSCLC. The central hypothesis is that identification of off-target-based anticancer mechanisms will reveal cancer-relevant targets and pathways providing novel personalized oncology opportunities with single drugs and drug combinations. The rationale for the proposed study is that novel targeted therapeutic options in NSCLC critically require predictive biomarkers and that mechanism-based biomarkers provide the greatest clinical value. To test this hypothesis, we will pursue the following specific aims: 1) Identify off-targets and -pathways that are modulated by kinase inhibitors; 2) Characterize the functional relevance of kinase inhibitor off-targets; and 3) Determine the potential for rational drug combinations based on identified off-targets. In Aim 1, our expertise in combining chemical and phosphoproteomics will be applied to provide a proteome-wide view of each kinase inhibitor's targets and signaling effects. In Aim 2, the functional relevance of inhibited targets and pathways will be determined by loss-of- function (RNAi, pharmacological inhibition) and rescue experiments (target overexpression, expression of drug- insensitive mutants). The translational potential will be investigated using primary tumor-derived cell lines and tissue samples to propose candidate biomarkers for future patient selection. Under Aim 3, compounds, which also target those off-target-relevant signaling pathways or which engage parallel pathways that, as evidenced by known driver mutations, are aberrantly activated in these cells, will be selected to examine potential synergistic interactions in vitro and ex vivo. The approach is innovative, because it represents a novel way of evaluating drug-induced phenotypic changes on the cellular level by specifically mining for and molecularly characterizing off-target effects that can be utilized for personalized oncology. The proposed research is significant as it will form the foundation for the discovery of mechanism-based biomarkers that enable successful clinical implementation of these novel therapeutic opportunities.